Defining life: the development of an artificial cell

A research team has provided a progress report on their attempts to construct …

Last year’s artificial cell was created by J. Craig Venter and colleagues using a "top-down" approach: they replaced the genome of a bacterium, Mycoplasma genitallium, with a synthetic DNA sequence they designed to contain the minimum set of genes required for life. It was an amazing feat, but all of the machinery necessary to make the cell work was already present within the bacterial shell. They simply hijacked it with their synthetic genome.

This year, an artificial cell project launched, and it intends to use a "bottom up" approach; Libchaber et al. plan to synthesize a viable cell from its basic components. They define these as the cell membrane, the border delineating the cell; the apparatus needed to coordinate metabolic activity; and finally the DNA, which acts as a both an information program driving metabolism and a code for remembering said program, much like a Turing tape. The hardest part, they think, will be getting these components to work with one another, as they describe in a progress report.

The membrane part is going to be easy. Phospholipids are amphipathic—their lipid tail is hydrophobic but their negatively charged phosphate group is hydrophilic—so phospholipid molecules spontaneously form sealed compartments in water. Self-assembling pore molecules were introduced to allow nutrients in and waste matter out of these compartments. However, these pore proteins have not yet been generated within a membrane using only internal gene expression.

The researchers note that “the construction of an artificial cell requires the development of an artificial environment.” The external medium needs to be much larger than the cell to allow for diffusion of waste products, and contain a finely tuned mixture of nucleotides to make RNA, amino acids to make proteins, and ATP for energy, among other components.

The cell will also need to transcribe DNA sequences to RNA, and translate that RNA to proteins. Cell-free transcription/translation extracts have been used by biologists for at least the past twenty-five years, and there have been improvements along the way. The first iteration consisted of cell extracts to provide the translational machinery, supplemented with a viral RNA polymerase for transcription. Later, the transcription machinery from E. coli was reconstituted from purified components to comprise what is known as the PURE system. When this is mixed with the viral RNA polymerase, you have a cell-free system where the entire composition is known.

The lack of the other cellular components present in these systems is not always a boon, however; sometimes proteins made using the PURE system don't fold properly, and additional proteins called chaperones need to be added to fix this problem.

Recently, this cell-free transcription/translation system has been put to use within the cell-sized phospholipid vesicles described earlier. So, it would seem we're well on our way to an artificial cell.

A few caveats at this point. First, it takes a bacterium one minute to make a protein form a moderate sized gene—from the start of transcription of the gene to a completely functional protein. Cell-free systems cannot begin to approach this rate, partially because they must operate at protein concentrations an order of magnitude lower than those in a cell. Moreover, we probably can't put all of the genes required for life under the control of a single viral polymerase—these polymerases are too limited to deal with a big enough genome. Finally, these systems do not have built-in systems for getting rid of old and potentially damaged RNAs and proteins.

Then there's the issue of genes. The Mycoplasma genitallium work demonstrated that about 200-400 genes are required to make a self-replicating cell. That's much too big to put on a typical DNA vector that we can manipulate. It's also too big for for a simple, always-on regulatory switch. The genome of bacteriophage Lambda, with thirty proteins, is possibly the most well-studied genetic network ever, but is still not completely understood.

To program a cell for self-replication—which, remember, is the goal here—the authors decided that combining small DNA subprograms would probably be best. Many of these programs are already available, notably those in the BioBricks Foundation. These programs were originally copied from living organisms, but they can be recombined in unique ways, and a few new ones have been generated. But the artificial life team acknowledges that the development of a DNA program sufficient to drive a cell is a significant hurdle.

Another major issue is coordinating the information contained in the DNA with activity of the nongenetic material. During cell division, for example, new membrane must be synthesized, and it must split when DNA is replicated to evenly partition the genetic information into the two daughter cells.

So, we've got a lot of hurdles before we get this thing to work. But we don't necessarily have to get it to work all that well at the start. An interesting aspect of the "bottom-up" approach is that it may allow us to better understand cellular evolution. The authors write that “assembling a synthetic cell unfolds the importance of physical aspects that are, in vivo, regulated by already evolved gene networks.” Building a cell from scratch could thus tell us what we really need, and what are just spandrels we got stuck with along the way, for good or ill.